Accurate and rapid counting of cellular elements in biological fluids is a necessity in the biomedical, pharmaceutical, and biological research fields. While there are many automated and semi-automated instruments for counting and examining cells, these instruments cannot measure low levels of cells (i.e., 100 cells/μL or less) or require pre-concentration or other time consuming steps to reach this detection level. Various biological fluids have a concentration of cells below 100 cells/μL and numerous procedures and protocols require that the low levels of cells in these fluids be measured. For instance, the current legal standards for transfusion products allow less than 20 white cells/μL in the United States and less than 4 white cells/μL in Europe before the products can be administered to humans. The concentration of both white cells and red cells in cerebrospinal fluid is less than 10 cells/μL. White and red cell counts are also performed when pleural, abdominal, and pericardial fluids are examined; generally, these cell levels are less than 50 cells/μL. Stem cell harvesting from donors cannot begin until there is a concentration of at least 10 cell/μL in the donor's peripheral blood. The presence of rare cells in various biological fluids may indicate cancer or other disease states. Generally these rare cells levels are 0.01 cells/μL or less. In addition to the difficulty in detecting low concentrations of cells, small sample size also presents a detection challenge. If only a few μL are available for assay, accurate enumeration of cells is difficult for many analytical systems. An apparatus and method to accurately measure the number of cells in biological fluids with a low or very low cell concentration or with low volumes would be beneficial.
While cell or particle counters are available, they do not accurately detect low levels of cells. For instance, presently commercially available automated cell or particle counters cannot measure cells levels below 500 to 1000 cells/μL. At a concentration of 10 cells/μL, the bias between manual and automated cell counting procedures has been reported be 999%. (Rabinovitch, A. and Cornbleet, P. J., Arch Pathol Lab Med, 118:13-17, 1994) In addition, fluidic elements used by these cell detection systems may be clogged up by viscous body fluids.
Flow cytometry methods may be employed to detect a low concentration of cells. However, large sample volumes are generally required in order for this process to be accurate. Flow cytometry systems achieve acceptable accuracy levels when 10,000 or more cells are counted; the error rate in samples having a concentration of 8 cells/μL has been reported to be 45%. (Dumont, L. J. and Dumont, D. F., Cytometry, 26:311-316, 1996) In a spinal fluid sample which contains 5 white cells/μL, a sample of 2 mL would be required in order for the system to detect 10,000 cells. A repeat second assay would also require 2 mL. In both pediatric and adult patients, the total volume of spinal fluid removed rarely exceeds 4 mL. Therefore, if flow cytometry were used to quantify the number of cells in a sample, all the fluid removed from the patient would have to be used for flow cytometry measurements; none would be available for the many other chemistry, microbiology, and cytology assays which are essential to spinal fluid procedures. Flow cytometers also encounter difficulty when viscous samples, such as body fluids, are analyzed because the samples may clog up the fluidics of the flow cytometer. Given these problems, flow cytometry may not be a practical solution to detecting low concentrations of cells.
A manual counting technique, employing a hemacytometer with either light or fluorescent microscopy may be used to count cells present in low concentrations. When a fixed-volume hemacytometer is used, a sample of biological fluid is diluted with a buffer-stain solution which keeps the cells intact and stains the cells so they are detectable. Either light or fluorescent microscopy is used (depending on the cell stain) to count the cells after a sample is loaded into the hemacytometer. A dilution calculation is used to determine the total number of cells per μL in the sample. This process is labor-intensive, time-consuming, and subject to human error.
Another drawback to using a hemacytometer is a lack of accuracy. The standard sample volume is 0.5 μL. Samples are usually diluted by a factor of two or more. Assuming a dilution factor of two, a sample with 10 cells/μL will have 5 cells/μL after dilution. If the sample volume is 0.5 μL, only 2.5 cells will be present in the hemacytometer. Given these low levels of cells, it is difficult to achieve accurate measurements.
The Nageotte hemacytometer overcomes some of the limitations of standard hemacytometers. The Nageotte hemacytometer has a sample volume of 50 μL and is used to measure the levels of white cells in transfusion products. A dilution factor of 10 using staining reagent is required in order to reduce background debris in the sample and make cell counting easier. As noted above, European regulations prohibit administration of a transfusion product that contains more than 4 white cells/μL. Given this limit, a liquid containing 4 cells/μL contains 0.4 cells/μL once it is diluted by a factor of ten. Since a 50 μL sample is examined in the Nageotte hemacytometer, 20 cells should be counted. However, studies indicate there is a 40% error rate for this procedure. (Rebulla, P. and Dzik, W., Vox Sang, 66:25-32, 1994).
A number of concentration procedures have been introduced to reduce the imprecision of low level cell counting methods by concentrating the cell levels prior to analysis. In one approach, 10 mL of a transfusion product is diluted with staining reagent. The diluted sample is centrifuged at 1000 g for 15 minutes. A second centrifugation at 300 g pellets the cells, allowing decanting of the supernatant. Cells in the concentrated sample volume are counted by manual and automated techniques. At cell counts below 1 cell/μL although the error rate was reported to range up to 36.2%. (Szuflad, P. and Daik, W. H., Transfusion, 37:277-283, 1997).
Various commercial procedures, designed to concentrate low levels of various biological fluids onto a slide for staining and subsequent cytopathology examination, are available. The CYTOFUGE™ 2 system from StatSpin, Inc. uses a disposable plastic cup fitted tightly against a standard glass slide. The sample to be concentrated is introduced into the cup and the device is then centrifuged. Cells are forced through the fluid onto the controlled small surface of the glass slide. The slide is then removed, stained, and examined. Similar systems are available from Wescor and Thermo Shandon.
The MonoPrep 2 system from MonoGen, Inc. uses a syringe assembly to concentrate cells onto a membrane. A plastic housing is attached to a standard 10 mL plastic syringe. The sample to be concentrated is aspirated into the syringe through the membrane. The cells are trapped onto the membrane as the liquid is drawn into the syringe. The membrane is then removed from the syringe housing and the cells transferred to a glass slide for cytopathology examination.
The prior art contains several other solutions to problems with filtration and concentration of samples. For instance, U.S. Pat. No. 5,252,293 discloses a slide with a porous membrane which can filter a sample at various locations on the slide. The slide and membrane provide a filter device with a capture surface for binding agents such as antibodies. The membrane is removed from a concentration device prior to analysis.
U.S. Pat. No. 5,308,483 discloses an in-line filter assembly where the filter membrane can be removed for identification or analysis of the material filtered from the fluid sample. U.S. Pat. No. 5,733,507 discloses a biological cell sample holder for use in infrared and/or Raman spectroscopy. A sample is added to the window of a sample holder, which selectively retains cells while the other components are filtered through the window. U.S. Pat. No. 5,484,572 is a fully-contained apparatus for collecting cells in fluid. A sample fluid is placed in a cup container. The cup container is pressurized and the fluid is forced through an outlet containing a specimen collection film. Specimen cells are captured on the film while the rest of fluid is filtered to another collection container. The specimen film may be removed for further analysis.
U.S. Pat. No. 5,240,861 discloses a device for concentrating liquid specimens which consists of a receptacle containing a membrane. The sample to be concentrated is placed in the receptacle on the upper surface of the membrane. A piston is screwed down toward the upper surface of the membrane, increasing pressure above the membrane and forcing small particles through the membrane. The sample is concentrated, for example, by having water removed from it. After the desired level of concentration is reached, the filtrate may be removed from the receptacle for further analysis.
None of the prior art discussed here discloses an apparatus that can concentrate a liquid specimen and subsequently allow automated cell enumeration in a single viewing.
None of the prior art discussed here shows a self-contained apparatus which prepares a sample for quantitative cell counting.
None of the prior art discussed above discloses a method to prepare a sample containing low levels of cellular elements for quantitative cell counting by standard imaging equipment.